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Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) in tetrahydrofolate binding.
Biochem J. 2009 Mar 15; 418(3):635-42.BJ

Abstract

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10-methylene THF. SHMT also catalyses several THF-independent side reactions such as cleavage of beta-hydroxy amino acids, transamination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect on either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.

Authors+Show Affiliations

Department of Biochemistry, Indian Institute of Science, Bangalore, India.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19046138

Citation

Pai, Vinitha R., et al. "Structural and Functional Studies of Bacillus Stearothermophilus Serine Hydroxymethyltransferase: the Role of Asn(341), Tyr(60) and Phe(351) in Tetrahydrofolate Binding." The Biochemical Journal, vol. 418, no. 3, 2009, pp. 635-42.
Pai VR, Rajaram V, Bisht S, et al. Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) in tetrahydrofolate binding. Biochem J. 2009;418(3):635-42.
Pai, V. R., Rajaram, V., Bisht, S., Bhavani, B. S., Rao, N. A., Murthy, M. R., & Savithri, H. S. (2009). Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) in tetrahydrofolate binding. The Biochemical Journal, 418(3), 635-42. https://doi.org/10.1042/BJ20081739
Pai VR, et al. Structural and Functional Studies of Bacillus Stearothermophilus Serine Hydroxymethyltransferase: the Role of Asn(341), Tyr(60) and Phe(351) in Tetrahydrofolate Binding. Biochem J. 2009 Mar 15;418(3):635-42. PubMed PMID: 19046138.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) in tetrahydrofolate binding. AU - Pai,Vinitha R, AU - Rajaram,V, AU - Bisht,Shveta, AU - Bhavani,B S, AU - Rao,N Appaji, AU - Murthy,M R N, AU - Savithri,H S, PY - 2008/12/3/pubmed PY - 2009/3/17/medline PY - 2008/12/3/entrez SP - 635 EP - 42 JF - The Biochemical journal JO - Biochem J VL - 418 IS - 3 N2 - SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10-methylene THF. SHMT also catalyses several THF-independent side reactions such as cleavage of beta-hydroxy amino acids, transamination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect on either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/19046138/Structural_and_functional_studies_of_Bacillus_stearothermophilus_serine_hydroxymethyltransferase:_the_role_of_Asn_341__Tyr_60__and_Phe_351__in_tetrahydrofolate_binding_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20081739 DB - PRIME DP - Unbound Medicine ER -